More than 48 members of the ABC transporter superfamily have been identified and three major subfamilies (ABCB, ABCC, and ABCG) are related to human multidrug resistance (MDR) and influence oral absorption and disposition of a wide variety of drugs, and as a result their expression levels have important consequences for susceptibility to drug-induced side effects, interactions, and treatment efficacy. The specific subclass members ABCB1 (MDR1/Pgp), ABCC1 (MRP1), and ABCG2 (BCRP) are known to significantly influence the efficacy of drugs and have unambiguously been shown to contribute to cancer multidrug resistance.1-2 Although a large number of compounds have been identified possessing ABC transporter inhibitory properties, only a few of these agents are appropriate candidates for clinical use as MDR reversing agents.3-4 Dual treatment with ABC transporter inhibitors in conjunction with chemotherapeutics is a common treatment strategy to circumvent MDR in cancers.5-6 However, the failure of current classes provide ample justification for identifying new classes of modulators and exploring the biology around them. These efflux pumps are expressed in many human tumors where they likely contribute to resistance to chemotherapy treatment. ABCB1, ABCC1, and ABCG2 are highly expressed in the gut, liver, and kidneys and they may restrict the oral bioavailability of administered drugs. ABCB1 and ABCG2 are also expressed in the epithelia of the brain and placenta and also in stem cells, where they perform a barrier function.7 More specifically, ABCG2 relevance as a clinical target has been well documented.8 This includes a mouse model using a human ovarian xenograft with Igrove1/T8 tumors,9 a system utilizing flavopiridol-resistant human breast cancer cells,10 an FTC/Ko 143 inhibition in vitro and mouse intestine model,11 and a phase I/II trial with lapatinib in glioblastoma multiforme.12 
Early clinical failures with ABCB1 inhibitors initially resulted in diminished enthusiasm. However, progress over the last decade has renewed activity in the field and a variety of modulators have been identified. ABC efflux transporter inhibition is now in its third generation with the majority of focus still on ABCB1. It has been observed that a large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps with numerous publications dedicated to the subject.13-16 A subset of these compounds will be discussed here. The first-generation of chemosensitizers were discovered from already approved drugs and included the calcium channel blocker verapamil (as well asnicardipine), cyclosporin A, and progesterone but dose-related toxicity and other adverse effects (i.e. solubility limitations) prevented progress into the clinic.17-24 Second and third generation inhibitors were drawn predominantly from the derivatization of first-generation molecules as well as from combinatorial chemistry targeted primarily at ABCB1. Some of the higher profile examples include:the cyclosporin A derivative valspodar (PSC-833)25; Vertex Pharmaceuticals' biricodar (VX-710)26-28; the anthranilamide based modulators XR905129, tariquidar (XR9576)30-32, XR957733-34, and WK-X-3434 35; the acridonecarboxamidederivativeelacridar (GF120918)36; the heteroaryloxypropanolamineszosuquidar (LY335979)37-39 and dofequidar (MS-209)40-41 (and the structurally related laniquidar (R101933)42-43), and diarylimidazoleontogen (OC144-093, ONT-093)44-47. The late generation inhibitors tended to be more potent and less toxic than the first-generation compounds, however, multiple issues remain.
Although much of the work to date is targeted at ABCB1, the selectivity profile of these inhibitors is significantly varied. Valspodar, tariquidar, elacridar, zosuquidar and ontogeny have been reported to be selective (though not necessarily specific) for ABCB1.25-28,30-31,40 Those specific for ABCC1 include the quinoline based MK571 and the uricosuric drug probenecide.48-49 Although there is significant progress with ABCB1 inhibitors, similar progress has not been made with ABCG2 inhibitors. The Aspergillusfumigatusmycotoxinfumitremorgin C (FTC) and its analogs Ko 132, Ko 134, and Ko 143 have been demonstrated to be selective inhibitors for ABCG2.11,50-52 Other imidazoline and β-carboline amino acid benzyl ester conjugates analogous to FTC were labeled ‘dual-acting’ due to a cytotoxicity that was coupled to their resistance reversing activity.53 Examples of cross pump inhibitors include verapamil, cyclosporin A, dofequidar, and reversanfor ABCB1/ABCC1 and biricodar and nicardipinefor ABCB1/ABCC1/ABCG2.28,54-56 
Structural information for all mammalian ABC transporter family members is relatively sparse, with ABCB1 being the most extensively studied. The presence of multiple, potentially overlapping, binding sites and possible interactions between them may account for diverse specificity of structurally and functionally unrelated modulators and substrates. This polyspecificity also raises questions as to which substrate should be used to demonstrate inhibitory potential of a new chemical entity. In order to understand the mechanism and to design more effective modulators, great effort has been made to study the interaction of substrates and modulators with these transporters.57 It was shown that most ABCB1 inhibitors are additionally substrates of the efflux pump.58 It is important to not only evaluate inhibitor potency in a given transporter, but also to profile its activity with other transporters as well as its interrelationship with substrate drugs. For instance, strong inhibition of ABCB1 by drugs like cyclosporine or verapamil in in vitro models proved to be limited in in vivo studies due to toxic pharmacological effects of the inhibitors.2 Our recent work further demonstrated differential cross-reactivity of inhibitors across ABCB1, ABCC1, and ABCG2 transporters and we demonstrated cross-reactivity of both these inhibitors across all three transporters, which could help explain such severe toxicity effects.56 Such interactions can be quite complex, since the array of substrate/non-substrate and inhibitor/non-inhibitor is further clouded by the possibility of multiple interaction sites and unwarranted cytotoxicity.
Several of the afformentioned small molecule inhibitors were selected to help profile the compounds of interest in vitro (FIG. 1). Compounds were chosen specifically for their reported selectivity profiles. The sub-micromolar modulator of ABCB1 XR9051 (a precursor to tariquidar) has been shown to reverse resistance to cytotoxic drugs such as doxorubicin and vincristine.3,29 The previously mentioned MK 571 is documented as a specific inhibitor of ABCC1.59 For direct comparison of selective inhibition of ABCG2 both FTC and Ko 143 were chosen.11,50-52 Also, the pyrazolopyrimidine reversan, with a similar core to our inhibitor class, was identified as an active inhibitor of ABCB1 and ABCC1.54 